Lung cancer is the leading cause of cancer mortality worldwide, with non-small cell lung cancer (NSCLC) the predominant histologic subtype of lung cancer and lung adenocarcinoma the major subset of NSCLC. Despite recent clinical progress with the use of specific targeted therapies, drug resistance remains a problem that limits patient survival. We propose a conceptually and technically innovative, multidisciplinary and collaborative project to improve the survival of lung cancer patients. We aim to capitalize on our recent discovery of the Hippo-YAP signaling pathway as a critical molecular circuit and therapeutic target in the many cancers driven by hyperactivation of RAS-RAF-MEK-ERK (RAS-MAPK) signaling, in which we have a long-standing interest. Resistance to RAF-MEK targeted therapy is a major clinical challenge in these cancers. RAF-MEK inhibitor treatment elicits a profound initial response in most BRAF mutant patients, but these responses are short-lived and some BRAF mutant and almost all RAS mutant patients fail to respond initially due to resistance. Through an unbiased genetic screen in BRAF mutant non-small cell lung cancer (NSCLC) cells, we discovered the Hippo pathway effector YAP acts as a parallel survival input to promote resistance to RAF-MEK inhibitor therapy. Combined YAP and RAF-MEK inhibition was synthetically lethal in several BRAF mutant tumor types and also in RAS mutant tumors. YAP promoted RAF-MEK inhibitor resistance in these tumors by increasing levels of the anti-apoptotic factor BCL-xL and suppressing levels of the pro-apoptotic factor BIM, intriguingly, in cooperation with MAPK signaling. Co-suppression of both YAP and MAPK signaling was required to suppress BCL-xL and increase BIM levels sufficiently to trigger apoptosis. Increased YAP was a biomarker of worse response to RAF-MEK inhibition in clinical samples with BRAFV600E, establishing the clinical relevance of our findings. These data reveal YAP as a novel mechanism of resistance to RAF-MEK targeted therapy and support co-suppression of YAP and MAPK signaling as a promising new therapeutic strategy. We propose to further test the hypothesis that YAP signaling is a critical molecular switch that regulates the biological and clinical response to targeted anti-cancer drugs, specifically in MAPK pathway driven NSCLCs, in 3 Specific Aims. In Aim 1, we will define the role of YAP in modulating targeted therapy response in key MAPK pathway driven NSCLC subsets, such as those with NF1 inactivation and EML4-ALK gene rearrangements (ALK+). We will further dissect the molecular function of YAP in resistance and basis of YAP-MAPK signaling crosstalk. In Aim 2, we will define a pharmacologic strategy to suppress YAP and enhance targeted therapy response, facilitating clinical translation. In Aim 3, we will define the role of YAP as a biomarker and target in NF1-altered and ALK+ NSCLC specimens, in addition to its role in BRAF/RAS mutant tumors established in our prior work. Overall, our project will offer new insight into the basis of therapy resistance and could improve patient survival.